Abstract
Membrane-protein interaction plays key roles in a wide variety of biological processes. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high-throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)-fusion proteins and a lipid containing a dark quencher, N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine (dabsyl-PE). The EGFP fluorescence emission intensity showed a large decrease (i.e., >50%) when EGFP-fusion proteins bound the vesicles containing 5 mol% dabsyl-PE. This simple assay, which can be performed using either a cuvette-based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains, including two pleckstrin homology domains, an epsin N-terminal homology domain, and a phox homology domain. The assay can also be applied to high-throughput screening of small molecules that modulate membrane binding of proteins.
Highlights
Membrane-protein interaction plays key roles in a wide variety of biological processes
Lack of high-throughput assay greatly hampers large-scale characterization of proteins to identify new and novel membrane binding proteins and screening of small molecule libraries to identify those molecules that can effectively modulate membrane binding of pharmacologically important proteins. To overcome these technical limitations and problems, we have developed a new sensitive and robust high-throughput membrane binding assay that is based on fluorescence quenching of enhanced green fluorescence protein (EGFP) fused to proteins by N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine incorporated in vesicles with various lipid compositions
As is the case with other fusion proteins, EGFPfusion proteins can be expressed in bacteria in high yield, as stable EGFP generally improves the expression yield of fusion partners, isolated protein domains and truncated proteins in particular
Summary
Membrane-protein interaction plays key roles in a wide variety of biological processes. The method offers many advantages over others, including high sensitivity, no requirement for protein labeling, and an ability to provide kinetic information It has limitations, such as Abbreviations: Akt1-PH-EGFP, C-terminal enhanced green fluorescence protein-tagged Akt plekstrin homology domain; dabsylPE, N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine; EGFP, enhanced green fluorescence protein; ENTH, epsin N-terminal homology; ENTH-EGFP, C-terminal enhanced green fluorescence protein-tagged epsin N-terminal homology domain; Ins(1,3,4,5)P4, inositol-1,3,4,5-tetrakisphosphate; NHERF1-PDZ2-EGFP, C-terminal enhanced green fluorescence protein-tagged second PDZ domain of NHERF1; PDK1-PH-EGFP, C-terminal enhanced green fluorescence proteintagged PDK1 plekstrin homology domain; PH, plekstrin homology; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; PS, phosphatidylserine; PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol3-phosphate; PtdIns(4)P, phosphatidylinositol-4-phosphate; PtdIns(5)P, phosphatidylinositol-5-phosphate; PtdIns(3,4)P2, phosphatidylinositol3,4-bisphosphate; PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; PtdIns(3,4,5) P3, phosphatidylinositol-3,4,5-trisphosphate; PtdInsP, phosphoinositide; PX, phox homology; SNX27-PX-EGFP, C-terminal enhanced green fluorescent protein-tagged sorting nexin 27 phox homology domain; SPR, surface plasmon resonance
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